Functional association of phosphoinositide‐3‐kinase with platelet glycoprotein Ibα, the major ligand‐binding subunit of the glycoprotein Ib–IX–V complex

Summary. Background: The adhesion receptor glycoprotein (GP)Ib–IX–V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet‐mediated tumor metastasis. The cytoplasmic C‐terminal domain of the ligand‐binding GPIbα subunit contains binding sites for filamin (residues 561–572, critically Phe568/Trp570), 14‐3‐3ζ (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide‐3‐kinase (PI3‐kinase) regulatory subunit, p85. Objectives: We previously showed that, as compared with wild‐type receptor, deleting the contiguous sequence 580–590 or 591–610, but not upstream sequences, of GPIbα expressed as a GPIb–IX complex in Chinese hamster ovary cells inhibited VWF‐dependent Akt phosphorylation, which is used as a read‐out for PI3‐kinase activity. Pulldown experiments using glutathione‐S‐transferase (GST)–p85 or GST–14‐3‐3ζ constructs, and competitive inhibitors of 14‐3‐3ζ binding, suggested an independent association of 14‐3‐3ζ and PI3‐kinase with GPIbα. The objective of this study was to analyze a further panel of GPIbα deletion mutations within residues 580–610. Results: We identified a novel deletion mutant, Δ591–595, that uniquely disrupts 14‐3‐3ζ binding but retains the functional p85/PI3‐kinase association. Deletion of other sequences within the 580–610 region were less discriminatory, and either partially affected p85/PI3‐kinase and 14‐3‐3ζ binding (Δ580–585, Δ586–590, Δ596–600, Δ601–605), or strongly inhibited binding of both proteins (Δ606–610). Conclusions: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14‐3‐3ζ in GPIb‐dependent signaling or platelet functional studies involving truncation of the C‐terminal residues in cell‐based assays and mouse models. The Δ591–595 mutation provides another strategy for determining the function of GPIbα‐associated 14‐3‐3ζ by selective disruption of 14‐3‐3ζ but not p85/PI3‐kinase binding.     

Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro

To cite this article: Banfi C, Brioschi M, Lento S, Pirillo A, Galli S, Cosentino S, Tremoli E, Mussoni L. Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro. J Thromb Haemost 2011; 9 : 1608–19. Summary. Background: Protease‐activated receptors (PARs) are G‐protein‐coupled receptors that function in hemostasis and thrombosis, as well as in the inflammatory and proliferative responses triggered by tissue injury. We have previously shown that PAR1 or PAR2 occupancy by specific PAR‐agonist peptides (PAR‐APs) induces tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs), where TF regulation by PAR1 (but not by PAR2) requires intact endothelial caveolin‐enriched membrane microdomains in which PAR1 and caveolin‐1 associate. Objectives: The aim of this study was to determine the effects of cholesterol‐lowering agents (statins) and cholesterol‐loading lipoprotein on PAR1‐AP‐mediated and PAR2‐AP‐mediated TF induction in HUVECs. Results: Statins completely prevented TF induction by PAR‐APs in an isoprenoid‐independent manner, induced the delocalization of PAR1 from caveolin‐enriched membrane microdomains without affecting PAR1 mRNA, and decreased PAR2 mRNA and protein levels. Statins also prevented PAR‐AP‐mediated extracellular signal‐related kinase 1/2 activation, which is crucial for TF induction. The redistribution of PAR1 is accompanied by the relocation of the membrane microdomain‐associated G‐protein α, caveolin‐1, and Src, which we previously showed to play a key role in signal transduction and TF induction. Conversely, cholesterol loading potently amplified PAR1‐AP‐induced TF, probably as a result of the increased abundance of PAR1 and the Src and G‐protein α signaling molecules in the caveolin‐1‐enriched fraction, without affecting PAR1 mRNA. Conclusions: As PARs have important functions in hemostasis, cancer, thrombosis, and inflammatory processes, our findings that statins prevent TF induction by PAR‐APs altering the membrane localization of PAR1 and the expression of PAR2 suggest that they may provide health benefits other than reducing atherosclerosis.     

Insect cold hardiness: the role of mitogen‐activated protein kinase and Akt signalling in freeze avoiding larvae of the goldenrod gall moth,Epiblema scudderiana

Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of cold hardiness to survive the winter. Here we report that protein kinase‐dependent signal transduction featuring mitogen‐activated protein kinase (MAPK) signalling cascades (extracellular signal regulated kinase, c‐jun N‐terminal kinase and p38 MAPK pathways) and the Akt (also known as protein kinase B, or PKB) pathway could be integral parts of the development of cold hardiness by E. scudderiana. We used Luminex technology to assess the protein levels and phosphorylation status of key components and downstream targets of those pathways in larvae in response to low temperature acclimation. The data showed that MAPK pathways (both total protein and phosphorylated MAPK targets) were inhibited after 5°C acclimation, but not ?15°C exposure, as compared with the 15°C control group. However, total heat shock protein 27 (HSP27) levels increased dramatically by ～12‐fold in the ?15°C acclimated insects. Elevated HSP27 may facilitate anti‐apoptotic mechanisms in an Akt‐dependent fashion. By contrast, both 5 and ?15°C acclimation produced signs of Akt pathway activation. In particular, the inhibitor phosphorylated Glycogen Synthase Kinase 3a (p‐GSK3) levels remained high in cold‐exposed larvae. Additionally, activation of the Akt pathway might also facilitate inhibition of apoptosis independently of GSK3. Overall, the current study indicates that both MAPK and Akt signal transduction may play essential roles in freeze avoidance by E. scudderiana.     

Atorvastatin or transgenic expression of TFPI inhibits coagulation initiated by anti‐nonGal IgG binding to porcine aortic endothelial cells

Summary. Background: Intravascular thrombosis remains a barrier to successful xenotransplantation. Tissue factor (TF) expression on porcine aortic endothelial cells (PAECs), which results from their activation by xenoreactive antibodies (Abs) to Galα1,3Gal (Gal) and subsequent complement activation, plays an important role. Objectives: The present study aimed to clarify the role of Abs directed against nonGal antigens in the activation of PAECs to express functional TF and to investigate selected methods of inhibiting TF activity. Methods: PAECs from wild‐type (WT), α1,3‐galactosyltransferase gene‐knockout (GT‐KO) pigs, or pigs transgenic for CD46 or tissue factor pathway inhibitor (TFPI), were incubated with naïve baboon serum (BS) or sensitized BS (with high anti‐nonGal Ab levels). TF activity of PAECs was assessed. Results: Only fresh, but not heat‐inactivated (HI), naïve BS activated WT PAECs to express functional TF. Similarly, PAECs from CD46 pigs were resistant to activation by naïve BS, but not to activation by fresh or HI sensitized BS. HI sensitized BS also activated GT‐KO PAECs to induce TF activity. TF expression on PAECs induced by anti‐nonGal Abs was inhibited if serum was pretreated with (i) an anti‐IgG Fab Ab or (ii) atorvastatin, or (iii) when PAECs were transgenic for TFPI. Conclusions: Anti‐nonGal IgG Abs activated PAECs to induce TF activity through a complement‐independent pathway. This implies that GT‐KO pigs expressing a complement‐regulatory protein may be insufficient to prevent the activation of PAECs. Genetic modification with an ‘anticoagulant’ gene (e.g. TFPI) or a therapeutic approach (e.g. atorvastatin) will be required to prevent coagulation dysregulation after pig‐to‐primate organ transplantation.     

Staphylococcus aureus‐induced complement activation promotes tissue factor‐mediated coagulation

Essentials

• Complement, Toll‐like receptors and coagulation cross‐talk in the process of thromboinflammation.
• This is explored in a unique human whole‐blood model of S. aureus bacteremia.
• Coagulation is here shown as a downstream event of C5a‐induced tissue factor (TF) production.
• Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation.

Summary

Background

There is extensive cross‐talk between the complement system, the Toll‐like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression.

Objectives

To study the relative roles of complement, TLRs and TF in Staphylococcus aureus‐induced coagulation.

Methods

Lepirudin‐anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF_{1 + 2}) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively.

Results

All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF_{1 + 2}, and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF_{1 + 2} levels to baseline levels.

Conclusions

S. aureus‐induced coagulation in human whole blood was mainly attributable to C5a‐induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus‐induced coagulation.     

Phosphoinositide 3‐kinase mediates CD40 ligand‐induced oxidative stress and endothelial dysfunction via Rac1 and NADPH oxidase 2

Summary. Objectives: CD40 ligand (CD40L) has been implicated as an inducer of reactive oxygen species (ROS) generation in endothelial cells, but definitive evidence for this and the in vivo relevance haves not been demonstrated fully. We thus investigated whether phosphoinositide 3‐kinase (PI3K) was linked to ROS generation and endothelial reactivity in response to CD40L. Methods and Results: CD40L treatment activated PI3K activity by regulating the association between PI3K p85 and the CD40 receptor. CD40L exposure also stimulated the GTPase Rac1, which is known to activate NADPH oxidases, and enhanced ROS formation, whereas PI3K inhibition or depletion by small interfering RNA (siRNA) prevented these responses. Subsequently, PI3K overexpression activated Rac1 and increased ROS generation. These responses were not observed in the presence of inactive Rac1 or siRNA against the NADPH oxidase subunit NOX2. Protein kinase Cζ mediates PI3K‐regulated NADPH oxidase activation by promoting cellular p47phox translocation. Importantly, PI3K inhibition prevented CD40L‐mediated ROS generation and endothelial dysfunction in a mouse model. In summary, PI3K mediates CD40L‐induced ROS production and subsequent endothelial dysfunction. Conclusions: Targeting PI3K may provide a new therapeutic approach in diseases associated with oxidative stress and endothelial dysfunction.     

Telmisartan‐enhanced hypercholesterolaemic serum‐induced vascular endothelial growth factor expression in immortalized human umbilical vascular endothelial cells

Objective. To clarify whether hypercholesterolaemia can increase vascular endothelial growth factor (VEGF) expression in human umbilical vascular endothelial cells (HUVECs) through the phosphatidylinositol 3‐kinase (PI3K) pathway, and whether a special angiotensin II receptor blocker, telmisartan, can attenuate VEGF expression induced by hypercholesterolaemia. Methods. Levels of VEGF expression, PI3K activity and angiogenesis in vitro were determined by various methods after HUVECs were incubated with hypercholesterolaemic serum or combined with telmisartan and/or wortmannin. Results. We found that hypercholesterolaemic serum (cholesterol ?0.08?mmol/L) can increase VEGF expression in HUVECs and that telmisartan cooperates with hypercholesterolaemic serum in promoting VEGF expression. The increased VEGF expression was associated with enhanced PI3K activity and could be significantly inhibited by wortmannin, a potent PI3K inhibitor. Likewise, hypercholesterolaemic serum significantly promoted angiogenesis in vitro, which could be inhibited when PI3K activity was suppressed. Conclusions. Our study suggests that hypercholesterolaemic serum induces VEGF expression through PI3K in HUVECs and that telmisartan cooperates with hypercholesterolaemia in promoting VEGF expression.     

Glycosylation of tissue factor is not essential for its transport or functions

See also Morrissey JH. Low‐carb tissue factor? This issue, pp 1508–10.DOI: 10.1111/j.1538‐7836.2011.04332.x . Summary. Background: Glycosylation plays an important role in protein function. The importance of glycosylation for tissue factor (TF) function is unclear. Objective: The aim of the present study is to investigate the importance of TF glycosylation in transport to the cell surface and its coagulant and signaling functions. Methods: Endothelial cells and peripheral blood mononuclear cells (PBMC) were treated with tunicamycin to inhibit N‐linked glycosylation. Site‐specific mutagenesis of one or more potential N‐linked glycosylation sites in TF was used to generate TF mutants lacking glycans. TF expression at the cell surface was determined in binding assays using ¹²⁵I‐FVIIa or ¹²⁵I‐TF mAb and confocal microscopy. TF coagulant activity was measured by factor (F) Xa generation assay, and TF signaling function was assessed by measuring cleavage of protease activated receptor 2 (PAR2) and activation of p44/42 MAPK. Results: Tunicamycin treatment reduced TF activity at the endothelial cell surface; however, this reduction was found to be the result of decreased TF protein production in tunicamycin‐treated cells. Tunicamycin treatment had no significant effect on TF activity or antigen levels in PBMC. No significant differences were observed in TF protein expression and procoagulant activity among cells transfected to express either wild‐type TF or TF mutants. A fully non‐glycosylated TF is shown to bind FVIIa and interact with FX with the same efficiency as that of wild‐type TF. Non‐glycosylated TF is also capable of supporting FVIIa cleavage of PAR2 and PAR2‐dependent p44/42 MAPK activation. Conclusions: Glycosylation is not essential for TF transport and coagulant or signaling functions.     

Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans: a ‘shear’ delight!

Jackson SP, Schoenwaelder SM. Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans: a ‘shear’ delight!J Thromb Haemost 2012; 10 : 2123–6.See also Nylander S, Kull B, Bjorkman JA, Ulvinge J‐C, Oakes N, Emanuelsson BM, Andersson M, Skarby T, Inghardt T, Fjellstrom O, Gustafsson D. Human target validation of phosphoinositide 3‐kinase (PI3K)β:effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor. This issue, pp 2127–36.     

Pre-germinated brown rice prevents high-fat diet induced hyperglycemia through elevated insulin secretion and glucose metabolism pathway in C57BL/6J strain mice

This study investigated the effect and mechanism of pre-germinated brown rice (PGBR) prevented hyperglycemia in C57BL/6J mice fed high-fat-diet (HFD). Normal six-week-old mice were randomly divided into three groups. Group 1 was fed standard-regular-diet (SRD) and group 2 was fed HFD for 16 weeks. In group 3, the mice were fed a HFD with its carbohydrate replaced with PGBR for 16 weeks. Comparing the SRD and HFD groups, we found the HFD group had higher blood pressure, higher concentrations of blood glucose and HbA1c. The HFD group had less protein expression of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3-kinase (PI3K), glucose transporter-4 (GLUT-4) and glucokinase (GCK) and greater expression of glucogen synthase kinase (GSK) in skeletal muscle. The HFD group also had less expression of IR, serine/threonine kinase PI3K-linked protein kinase B (Akt/PKB), AMP-activated protein kinase (AMPK), GCK and peroxisome proliferator-activated receptor γ (PPARγ) in liver. In the HFD + PGBR group, the PGBR could reverse the disorders of blood pressure, blood glucose, HbA1c and increase insulin concentration. PGBR increased the IR, IRS-1, PI3K, Akt, GLUT-1 and GLUT-4 proteins, and ameliorated AMPK, GCK, GSK and PPARγ proteins. Together, PGBR prevented HFD-induced hyperglycemia through improving insulin levels, insulin receptor, glucose transporters and enhancing glucose metabolism.     

Erythropoietin attenuates hypertrophy of neonatal rat cardiac myocytes induced by angiotensin‐II in vitro

Objective: Erythropoietin (EPO) is a haematopoietic hormone that has been confirmed as a novel cardioprotective agent. In this study, we test the hypothesis that EPO inhibits angiotensin‐II (Ang‐II)‐induced hypertrophy in cultured neonatal rat cardiomyocytes. Material and methods: Cultured neonatal rat cardiomyocytes were used to evaluate the effects of EPO on Ang‐II‐induced hypertrophy in vitro. The surface area and mRNA expression of atrial natriuretic (ANF) myocytes were employed to detect cardiac hypertrophy. A phosphatidylinositol 3′‐kinase (PI3K) inhibitor LY294002 and an endothelial nitric oxide synthase (eNOS) inhibitor l‐NAME were also employed to detect the underlying mechanism of EPO. Intracellular signal molecules, such as Akt (PKB), phosphorylated Akt, eNOS and transforming growth factor‐β1 (TGF‐β1) protein expression were determined by Western blot. Nitric oxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed using an NO assay kit. Results: The results indicate that EPO significantly attenuates Ang‐II‐induced hypertrophy shown as inhibition of increases in cell surface area and ANF mRNA levels. NO production was also increased proportionally in the EPO‐treated group. EPO enhanced Akt activation and eNOS protein expression, whereas LY294002 or l‐NAME partially abolished the anti‐hypertrophic effect of EPO, accompanied by a decrease in Akt activation, eNOS protein expression and/or a reduction of NO production. EPO also down‐regulated the protein expression of TGF‐β1. Conclusion: We conclude that EPO attenuates cardiac hypertrophy via activation of the PI3K‐Akt‐eNOS‐NO pathway and the down‐regulation of TGF‐β1.     

The PKB inhibitor Akti‐1/2 potentiates PAR‐1‐mediated platelet function independently of its ability to block PKB

Summary. Background: The role of PKB in platelet function is poorly defined due to the lack of genuinely selective small‐molecule inhibitors and limiting genetic models. Recently, a selective, non‐ATP‐competitive PKB inhibitor, Akti‐1/2 has been reported, but the efficacy and specificity against PKB activation in platelet function is unknown. Objective and methods: To determine the effect of the PKB inhibitor Akti‐1/2 on PKB activation and platelet function by Western blotting and aggregometry/flow cytometry, respectively. Results: Akti‐1/2 potently inhibited thrombin‐mediated PKB phosphorylation on Thr³⁰⁸ and Ser⁴⁷³ and phosphorylation of its downstream substrate GSK3β, with a negligible effect on the phosphorylation of pleckstrin, p38, ERK and JNK. Surprisingly, Akti‐1/2 strongly potentiated PAR‐1‐mediated platelet aggregation. This effect persisted in the presence of PI3 kinase inhibitors, indicating a mechanism of action that is independent of PKB. Potentiation of aggregation by Akti‐1/2 was associated with increased [Ca⁺⁺]_i, PKC activation and degranulation and was ablated by agents that antagonized this pathway. Conclusions: The PKB inhibitor Akti‐1/2 increases PAR‐1‐mediated platelet responses in a PKB‐independent, Ca⁺⁺/PKC‐dependent manner. This effect is strong and rapid and may impact on the therapeutic application of Akti‐1/2 and structurally related compounds.     

The Syk‐kinase inhibitor R406 impairs platelet activation and monocyte tissue factor expression triggered by heparin‐PF4 complex directed antibodies

Summary. Background: Heparin‐induced thrombocytopenia (HIT) is a rare but severe complication of heparin therapy in which immunoglobulin G (IgG) antibodies against the platelet factor 4–heparin complex activate platelets through the FcγRIIA receptor. Clustering of FcγRIIA initiates signaling cascades involving tyrosine kinases including the spleen tyrosine kinase (Syk). Moreover, besides the critical role of platelets, the expression of tissue factor (TF) by human monocytes triggered by HIT antibodies has been shown to contribute to the hypercoagulability and the thrombotic complications in HIT patients. Objectives: We investigated the effect of R406, a small molecule inhibitor of Syk developed as a potential treatment of autoimmune diseases, allergic disorders and B‐cell related hematological malignancies, on FcγRIIA‐mediated platelet activation. To further assess the potential activity of Syk inhibitors in HIT treatment, the effect of R406 was also evaluated on HIT antibodies‐induced expression of TF and procoagulant activity of monocytic cells. Results: We show that R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross‐linking by specific antibodies or by sera from HIT patients. Syk inhibition efficiently prevents FcγRIIA‐induced LAT phosphorylation and activation of phosphoinositide 3‐kinase, Akt, phospholipase Cγ2 and p38 MAP‐kinase. As a consequence, FcγRIIA‐induced platelet aggregation, granule secretion and microparticles production are strongly inhibited by R406. Moreover, the Syk inhibitor efficiently impairs the expression of TF and the procoagulant activity of human monocytes triggered by HIT antibodies. Conclusion: Syk inhibitors may be of therapeutic interest in the treatment of HIT by reducing HIT antibodies‐mediated platelet activation and monocyte procoagulant activity.     

Human target validation of phosphoinositide 3‐kinase (PI3K)β: effects on platelets and insulin sensitivity,using AZD6482 a novel PI3Kβ inhibitor

Nylander S, Kull B, Björkman JA, Ulvinge JC, Oakes N, Emanuelsson BM, Andersson M, Skärby T, Inghardt T, Fjellström O, Gustafsson D. Human target validation of phosphoinositide 3‐kinase (PI3K)β:effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor. J Thromb Haemost 2012; 10: 2127–36. See also Jackson SP, Schoenwaelder SM. Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans – a ‘shear’ delight! This issue, pp 2123–6. Summary. Background: Based on in vitro and animal data, PI3Kβ is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. Objective: To strengthen the PI3Kβ target validation using the novel, short‐acting inhibitor AZD6482. Methods and results: AZD6482 is a potent, selective and ATP competitive PI3Kβ inhibitor (IC₅₀ 0.01 μm ). A maximal anti‐platelet effect was achieved at 1 μm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti‐thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3‐h infusion. The ex vivo anti‐platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin‐induced human adipocyte glucose uptake in vitro (IC₅₀ of 4.4 μm ). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 μm but reduced by about 60% at a plasma exposure of 27 μm . In man, the homeostasis model analysis (HOMA) index increased by about 10–20% at the highest plasma concentration of 5.3 μm . Conclusions: This is the first human target validation for PI3Kβ inhibition as anti‐platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at ‘supratherapeutic’ plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.     

Protein kinase C alpha and beta are positive regulators of thrombus formation in vivo in a zebrafish (Danio rerio) model of thrombosis

Summary. Background: The zebrafish (Danio rerio) is becoming an attractive model organism for the assessment of gene function in thrombosis in vivo. Zebrafish, as a thrombosis model, have several advantages, with the capacity to follow thrombus formation at high resolution in real time using intravital microscopy, without the need for complex surgical techniques, and the capability to rapidly knockdown gene expression using morpholino antisense approaches. Objectives: We have recently shown, in mouse models, that protein kinase C alpha (PKCα) plays a critical role in regulating thrombus formation in vivo. PKC beta (β) plays a non‐redundant role also in platelet function in vitro, but the function of this gene had not yet been assessed in vivo. Methods: In the present study, we analyzed the function of both PKCα and PKCβ in the zebrafish model in vivo, by live imaging using a laser‐induced injury of the main caudal artery in 3‐day‐old larvae. Results: We showed that D. rerio express orthologs of both the PKCα and PKCβ genes, with high sequence identity. Translation blocking and splice‐blocking morpholinos effectively and specifically knockdown expression of these genes and knockdown with either morpholino leads to attenuated thrombus formation, as assessed by several quantitative parameters including time to initial adhesion and peak thrombus surface area. Conclusions: Our data indicate that these two highly related genes play non‐redundant roles in regulating thrombosis, an observation that supports our previous in vitro murine data, and suggests unique roles, and possibly unique regulation, for PKCα and PKCβ in controlling platelet function in vivo.     

No evidence for the presence of tissue factor in high‐purity preparations of immunologically isolated eosinophils

Summary. Background: To date, there is no unequivocal opinion on whether human eosinophils express tissue factor (TF). Therefore, we studied the expression of TF protein and activity in resting or stimulated immunologically purified human eosinophils. Methods and results: By use of immunologic isolation, we achieved over 96% purity of eosinophil preparations, and contamination by CD14‐positive cells was below 0.3%. Flow cytometric [fluorescence‐activated cell sorting (FACS)] analysis of eosinophils revealed no surface expression of TF antigen in resting or stimulated eosinophils. Immunoblotting of eosinophil lysates did not show any TF protein under resting or stimulated conditions. The lysates of resting or stimulated eosinophils contained no detectable levels of TF procoagulant activity. In contrast, monocytes, stimulated in plasma or medium, possessed readily detectable TF levels on the cell surface and in cell lysates as detected by FACS and immunoblotting. This was active TF antigen, as confirmed by TF activity assay (19.2 ± 4.2 and 28.6 ± 3.1 mU per 10⁶ cells, stimulated in medium or plasma, respectively). We found no detectable TF mRNA levels in resting or stimulated eosinophils by real‐time polymerase chain reaction (PCR), whereas in monocytes TF mRNA levels were significantly increased after stimulation. Conclusions: Our data indicate that there is no evidence for TF expression in high‐purity preparations of immunologically isolated eosinophils.